Several alphaviruses are significant human pathogens, and are among the primary causes of mosquito-borne viral encephalitis in the Americas. Replication of alphaviruses in cells of the vertebrate host has been studied extensively, and the roles that the viral glycoproteins play in the replication cycle, pathogenesis, virulence, and immune recognition have been defined in detail. Information from such studies has been instrumental for the design of live attenuated alphavirus vaccines. In contrast, relatively little is known about the molecular determinants that govern viral growth in mosquito cells. Still less is known about the viral and host determinants that influence virus spread throughout the mosquito, or that influence virus transmission by this vector to susceptible vertebrate hosts. Information in these areas will be necessary for the design and implementation of novel strategies that view the mosquito vector as a target for intervention in the natural alphavirus maintenance cycle. The long term goal of our studies is to characterize the fundamental structural and functional properties of alphavirus glycoproteins during viral replication in cells of the arthropod host. The specific goal of the proposed studies is to characterize the molecular basis for an arthropod cell-specific host restriction displayed by a well characterized class of alphavirus mutants. In addition, we will investigate the relevance of this cell culture based phenotype to viral replication in vivo, by assessing phenotypes of virus replication, dissemination, tissue tropism, and reversion within the intact vector following infection of Aedes albopictus mosquitos by natural feeding or by intrathoracic inoculation. These studies will use a wild-type parental strain of Sindbis virus (TRSB) and several well characterized TRSB-derived mutants that are defective for cleavage of the PE2 precursor glycoprotein, and which retain PE2 in place of E2 in mature virions. The PE2-cleavage defect manifest by these viruses has been linked to phenotypic alterations in viral virulence, pathogenesis, and host-range. Each of these phenotypes are linked to aberrant viral glycoprotein functions that appear to be required for virus maturation and which are acutely influenced by the carbohydrate processing phenotypes of the host cell.